ARTICLE. Introduction. Mónica M. Steciow 1*, Enrique Lara 2 *, Christophe Paul 2, Amandine Pillonel 2, and Lassaad Belbahri 2

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1 doi:0.5598/imafungus IMA Fungus 5(2): (204) Multiple barcode assessment within the Saprolegnia-Achlya clade (Saprolegniales, Oomycota, Straminipila) brings order in a neglected group of pathogens Mónica M. Steciow *, Enrique Lara 2 *, Christophe Paul 2, Amandine Pillonel 2, and Lassaad Belbahri 2 Instituto de Botánica Spegazzini, Facultad de Cs. Naturales y Museo, Universidad Nacional de La Plata, 53 N 477, (900) La Plata, Buenos Aires, Argentina. 2 Laboratory of Soil Biology, Department of Biology, University of Neuchâtel, Rue Emile Argand, CH-2000 Neuchâtel, Switzerland; corresponding author lassaad.belbahri@unine.ch *Both these authors contributed equally to the work. Abstract: The Saprolegnia-Achlya clade comprises species of major environmental and economic importance due to their negative impact on aquaculture and aquatic ecosystems by threatening fishes, amphibians, and crustaceans. However, their taxonomy and phylogenetic relationships remain unresolved and suffer from many inconsistencies, which is a major obstacle to the widespread application of molecular barcoding to identify pathogenic strains with quarantine implications. We assessed phylogenetic relationships of major genera using three commonly used markers (ITS, SSU rrna, and LSU rrna). A consensus tree of the three genes provided support for nine clades encompassing eleven documented genera and a new clade (SAP) that has not been described morphologically. In the course of this study, we isolated a new species, Newbya dichotoma sp. nov., which provided the only culture available for this genus. In parallel, we attempted to summarize the evolution of traits in the different genera, but their successive reversals rendered the inference of ancestral states impossible. This highlights even more the importance of a bar-coding strategy for saprolegniacean parasite detection and monitoring. Key words: barcoding ITS LSU rrna morphology Newbya SSU rrna water moulds zoospore discharge Article info: Submitted: 30 April 204; Accepted: 24 November 204; Published: 0 December 204. Introduction Oomycetes (or water moulds) are protists with a mycelial growth that were traditionally classified within Fungi, and that have been relocated within Heterokonta, also known as Straminipila, based on their molecular phylogeny. They are ubiquitous in freshwaters, but also in soils and in marine environments, where they can be found either as parasites or free-living. Saprolegnia-Achlya is a monophyletic clade of mainly freshwater species that forms a sister clade to Aphanomyces and includes notorious animal pathogens. These organisms are abundant in the environment, where they behave as destructive pathogens in fish (Phillips et al. 2008), Daphnia (Wolinska et al. 2009), insects (Pelizza et al. 20), amphibians (Fernández-Beneítez 20), and crayfish (Krugner-Rigby et al. 200). As such, they are responsible for millions of dollars losses to the worldwide aquaculture industry (van West 2006), with considerable damage to highly valued fish such as salmonids (Hussein & Hatai 999). They can also threaten endangered wildlife, and cases of local extinctions of amphibian populations due to members of this group have been reported (Bragg 958, 962), as well as declines of wild fish stocks (van West 2006). It is therefore of crucial importance to detect these organisms and identify them quickly and reliably, in order to apply quarantine measures (Lara & Belbahri 20). General strategies for detecting these parasites rely on isolation and cultivation of strains isolated from infected animals followed by morphological identification, a strategy that is time-consuming and requires highly trained specialists. As morphological features used for identification of species and genera require the presence of reproductive and disseminative structures which appear sometimes only after weeks (Steciow 2003), urgent measures cannot be taken early enough to prevent epidemics. For this reason, a molecular barcoding approach could bring a valuable tool for quickly and accurately monitoring disease outbreaks prior to taking appropriate measures. However, this approach is dependent on the existence of a reliable database that allows confident assignment of pathogenderived or environmental sequences to known species. In particular, a well-documented molecular systematics study can provide a base for identifying known species and placing newly discovered strains. Unfortunately, existing databases suffer major drawbacks caused by: () taxonomic inconsistencies; and (2) misidentified strains sent to and preserved in public collections of fungus cultures. It is therefore urgent to clarify the taxonomy within this group 204 International Mycological Association You are free to share - to copy, distribute and transmit the work, under the following conditions: Attribution: You must attribute the work in the manner specified by the author or licensor (but not in any way that suggests that they endorse you or your use of the work). Non-commercial: You may not use this work for commercial purposes. No derivative works: You may not alter, transform, or build upon this work. For any reuse or distribution, you must make clear to others the license terms of this work, which can be found at Any of the above conditions can be waived if you get permission from the copyright holder. Nothing in this license impairs or restricts the author s moral rights. volume 5 no

2 Steciow et al. by providing a reliable phylogenetic framework based on sequence information and morphological features. In addition, it is useful to address inconsistencies that limit the use of culture collection resources and propagate errors in subsequent studies. Several species have been described but later assigned to either Achlya or Saprolegnia. These two genera are traditionally distinguished by the mode of discharge of the zoospores; in Saprolegnia flagellate stages encyst to give rise to a second flagellated form (diplanetism), whereas in Achlya the released zoospores are the only dispersive form (monoplanetism) (Daugherty et al. 998, Johnson et al. 2002). However, these two gross types have several subtypes, and species identification can only be achieved using a combination of characters. In addition, these characters can only be assessed after the generation of sexual and asexual (dispersive) forms, which requires the testing of many different culture conditions and long incubation periods, a time-consuming process that can be precious in the case of a disease outbreak. Therefore, development of a robust barcoding strategy to identify possible pathogens or implement quarantine measures would be extremely useful. In order to overcome these caveats, we recompiled the morphological features used to identify species of Saprolegniaceae. In parallel, we performed phylogenetic analyses of the group based on the three most frequently used markers, ITS, SSU and LSU rrna, and compared their respective resolution powers. We also inferred a consensus tree and inferred possible scenarios for trait evolution. And, finally, we described a new species from a little-known genus, Newbya dichotoma sp. nov. isolated from floating organic matter associated with fishpond water. Materials and Methods Isolation of Newbya dichotoma Floating organic matter (twigs, leaves) were collected from the Chimehuin River (Neuquén Province, Argentina) and taken to the laboratory in separate sterile polyethylene bags. The methods for collection and isolation of mycelium-forming organisms with a flagellated stage described by Coker (923), Johnson (956), Sparrow (960), and Seymour & Fuller (987) were used. Baits were placed in water culture in sterilized Petri dishes containing several halves of hemp seeds (Cannabis sativa) and incubated at room temperature (5 20 C). When mycelial growth was observed on the seeds, a single hypha was isolated and transferred to cornmeal-agar medium (CMA) to obtain an axenic culture. After 3 4 d, a block of agar from the edge of each colony was cut off and placed in sterilized Petri dishes containing water. Several preparations were made for each sample and zoosporic organisms were identified using the vegetative organs (shape and size of the hyphae), asexual structures (shape of zoosporangium and spores), and sexual organs (structure of the oogonium and antheridium). Observations and measurements were made with an Olympus BX 40 microscope (Olympus Optical, Tokyo) equipped with phase contrast optics. During our investigations this new species was repeatedly isolated from the same locality in May and June 2009, and May 200. DNA extraction, PCR, sequencing and phylogenetic analyses Reference strains used in this study were obtained from CBS- KNAW Fungal Biodiversity Centre (Utrecht, The Netherlands) and VKM (All-Russian Collection of Microorganisms, Moscow, Russian Federation) and are listed in Table. DNA was extracted with a guanidine thiocyanate buffer protocol as in Lara & Belbahri (20). PCR was performed using the wide-spectrum primers ITS4 and ITS6 (White et al. 990) for the ITS region, EK 42F and EK 498R for SSU rrna gene (López García et al. 200), and ITS4 and 28S-564R as described in Steciow et al. (203). The PCR products were sequenced with an ABI PRISM 3700 DNA Analyzer (PE Biosystems, Geneva) using a BigDye TM Terminator Cycle Sequencing Kit (PE Biosystems). Sequences have been deposited in GenBank with accession numbers provided in Table. Sequences from representatives from all genera from the Saprolegnia-Achlya clade present in GenBank for the three marker genes have been surveyed and collected to complete the alignments that generated the trees presented here. Some sequences from the sister-clade Aphanomyces were added as outgroup. Although several diverging nomenclatures were used by different sequence submitters, we kept original names as they were. The nomenclature used in this study is that proposed by Dick (200) and Spencer et al. (2002). Sequences were aligned manually using BioEdit software (Hall 999). The phylogenetic trees were reconstructed using a Maximum Likelihood approach as implemented in MEGA v. 6 (Tamura et al. 203) using the following parameters: general time reversible model with invariant sites and four categories among site variation, and 000 bootstraps to evaluate node robustness. Alignments for the three genes comprised respectively 559 bp for ITS region, 633 bp for SSU rrna gene and 785 bp for the LSU rrna gene. Trees were rooted with sequences derived from Aphanomyces species and relatives, as these organisms are the closest known relatives of the Saprolegnia-Achlya clade (Lara & Belbahri 20). Because publicly available sequences from different gene markers did not derive from the same organisms, we could not concatenate the three markers and proceed to a joint phylogenetic analysis. Therefore, we inferred a consensus tree on the basis of keeping every node that was supported robustly by at least one marker and not contradicted by another. Results Gene trees and consensus tree The tree based on the ITS region showed a robust support for the monophyly of all genera except Achlya, with bootstrap supports ranging between 0.89 and (Fig. ). The genus Leptolegnia appears paraphyletic if Geolegnia is accepted as a separate genus; likewise, Thraustotheca appears nested within Achlya, turning the latter paraphyletic too. Deeper branchings (i.e. relationships between genera) are generally not supported, except for SAP and Leptolegnia/Geolegnia (bootstrap value = 0.97) and Aplanes/Aplanopsis/Newbya (bootstrap value = ). This tree placed Newbya, at the base of the genera Aplanes and Aplanopsis. This branching 440 ima fungus

3 Barcode assessment within the Saprolegnia-Achyla clade Table. List of the cultures analyzed in this study, including the CBS accession number and GenBank accession numbers for each gene sequenced. Species VKM, LPSC and CBS GenBank accession numbers accession numbers ITS SSU LSU Achlya debaryana VKM F-904 KP KP09837 KP Achlya sparrowii VKM F-227 KP KP KP Aplanopsis spinosa CBS KP KP KP Aplanes treleaseanus VKM F-229 AB29373 KP KP Aplanopsis spinosa CBS KP KP KP Leptolegnia caudata CBS 343 KP09834 KP ND Leptolegnia caudata CBS KP KP KP Leptolegnia sp. CBS KP KP ND Protoachlya paradoxa CBS KP09835 KP ND Isoachlya humphreyana CBS 0057 KP KP ND Pythiopsis intermedia CBS KP KP ND Pythiopsis terrestris CBS 0058 KP KP KP Pythiopsis terrestris CBS 0059 KP KP ND Thraustotheca clavata CBS KP KP HQ66523 Leptolegnia caudata CBS KP KP ND Thraustotheca clavata CBS KP KP HQ Newbya dichotoma LPSC 877 KP KP KP09836 ND = Non determined. Table 2. Morphological features of the Saprolegnia-Achlya clade genera including discharge mode, sporangial characteristics and zoospore motile flagellate phases. Genus Discharge mode Sporangia Flagellated zoospores Saprolegnia saprolegnoid New sporangia renewed by internal proliferation dimorphic (primary+secondary) Pythiopsis saprolegnoid New sporangia renewed sympodially,in cymose or monomorphic (primary) basipetalous fashion Protoachlya protoachloid Some zoospores swim and some encyst and form a loose dimorphic (primary +secondary) cluster Isoachlya saprolegnoid New sporangia formed by cymose branching or internal dimorphic (primary+ secondary) proliferation Scoliolegnia saprolegnoid New sporangia renewed by internal proliferation, cymose dimorphic (primary +secondary) branching or basipetal succession Newbya achlyoid Sporangia fusiform or filiform, renewed by cymose branching monomorphic (secondary) Aplanes aplanoid or achlyoid Sporangia cylindrical or fusiform; spores germinate in situ, 0 lack flagellate zoospores inside sporangium Calyptralegnia thraustothecoid or Sporangia fusiform, cylindrical or clavate; renewed sympodially, monomorphic (secondary) calyptralegnoid or in a basipetalous or cymose fashion Aplanopsis achlyoid or without Sporangia usually unknown no planonts were seen sporangia Achlya achlyoid New sporangia renewed sympodially, in cymose or basipetalous monomorphic (secondary) fashion Thraustotheca thraustothecoid Sporangia clavate, obpyriform, fusiform, renewed sympodially monomorphic (secondary) or in cymose manner Leptolegnia leptolegnoid Sporangia cylindrical, elongate, sometimes renewed by internal dimorphic (primary+ secondary) proliferation, spores produced in a single row Geolegnia geolegnoid Sporangia cylindrical, elongate or swollen at intervals, 0 lack flagellate zoospores nonseptate, formed sympodially, spores produced in a single row Dictyuchus dictyucoid Sporangia cylindrical to clavate, renewed sympodially or in basipetalous fashion, spores produced in a or more than one row monomorphic (secondary) Brevilegnia achlyoid, dictyucoid or brevilegnoid Sporangia cylindrical to clavate, renewed sympodially, in cymose or basipetalous fashion, spores produced in a or more than one row monomorphic or not swimming Aphanomyces achlyoid Sporangia filamentous, spores produced in a single row dimorphic (primary lack flagella) volume 5 no. 2 44

4 Steciow et al Protoachlya sp. Argentina 3. EU5549 strain UNCW244 DQ39356 Protoachlya paradoxa ATCC DQ «Pythiopsis cymosa» CBS (*) strain NT GU04277 strain EF75575 strain EM32A GU04278 Leptolegnia caudata CBS 343 «Thraustotheca clavata» CBS (*) strain BBE5 EU24749 strain H2E3 EU2475 strain AE EU24752 strain BBE20 EU24753 strain SP GU04279 strain HE EU24754 strain H3E3 EU24760 strain BB08 EU strain H3E5 EU24757 strain H3E EU24756 strain H3E2 EU24755 strain P8203 GU strain P8203 FJ80989 strain P8205 GU Leptolegnia caudata CBS Geolegnia helicoides KF Leptolegnia sp. CBS strain SAP_RIM0 FJ79490 strain SAP_ZEL03+04 FJ strain SAP_SEC0 FJ strain SAP_TNR0 FJ strain SAP_ZLU0 FJ strain SAP_ZEL0 FJ strain SAP_BRN0 FJ strain SAP_VIR0 FJ strain SAP_ZEL03+04 FJ strain SAP ZEL03+04 FJ strain VI03659 AM strain CBS AY strain VI03660 AM94703 strain SAP3_ZLU0 FJ79490 strain SAP3_LAB0 FJ strain VI03839 AM strain WM_3 EU54492 Scoliolegnia asterophora CBS AB29398 strain BBE7 AB strain WM_2 EU5449 strain EU Isoachlya anisospora EU24748 Isoachlya anisospora EU24086 Isoachlya anisospora EU24746 Isoachlya anisospora EU24747 Isoachlya humphreyana CBS 0056 AB29382 Isoachlya humphreyana CBS 0057 Isoachlya eccentrica CBS 006 AB29376 Isoachlya Isoachlya anisospora CBS 0060 AB29384 Isoachlya anisospora EU24004 Protoachlya Leptolegnia Geolegnia SAP Scoliolegnia Pythiopsis terrestris CBS 0058 strain AB29375 Pythiopsis terrestris CBS 0058 AB29383 Pythiopsis terrestris CBS 0059 Pythiopsis Pythiopsis intermedia CBS Pythiopsis cymosa CBS 0055 AB2938 Achlya radiosa AF2860 Achlya sparrowi VKM F-227 Achlya colorata AF2859 Achlya racemosa AF Achlya racemosa Argentina 3.3 EU5550 Achlya I strain CBS AY30498 Thraustotheca strain CBS0065 HQ64404 Thraustotheca clavata CBS Thraustotheca clavata CBS Thraustotheca sp. EU555 Thraustotheca sp. AY64789 strain AY64789 strain S3 AY Achlya debaryana VKM F strain EU84969 strain AY64796 Achlya II Achlya bisexualis DQ40320 Achlya bisexualis EU strain EU84968 Achlya crenulata AF Aplanopsis spinosa CBS Aplanopsis spinosa CBS Aplanopsis strain AB29373 Aplanes papillosus AF286 Aplanes androgynus AB29374 Aplanes Aplanes treleaseanus VKM F Newbya oligacantha AF Newbya stellata EU24762 Newbya Newbya dichotoma spec. nov. Saprolegnia anomalies DQ strain EU5230 Saprolegnia ferax GQ9935 strain ATCC DQ strain BP_S AY strain WD4C EF26335 strain WDJ EU240 strain WDG EU24008 strain WDM EU2404 strain WD3D EU24020 strain WD3G EU24022 strain WD3A_EU24028 strain. WD3B EU24029 strain SAP2 FJ Saprolegnia strain W805b EF52546 Saprolegnia bulbosa AY2670 Saprolegnia longicaulis K07 EU Saprolegnia salmonis EU5553 strain UNCW27 DQ39353 Saprolegnia parasitica AM strain ATCC 5272 AY64788 Saprolegnia australis GQ99078 strain CBS AB29394 strain WM_ EU54490 strain ATCC 9025 AY strain AY64795 strain PCRTB2 GU04269 strain O3EG GU0427 strain RACL2005 EU strain CBS AY30502 Saprolegnia kaufmanniana EU24766 strain EM4 EU34837 strain SCAAD GU strain UNCW289 DQ strain UNCW299 DQ strainuncw298 DQ strain UNCW29 DQ Saprolegnia litoralis AM strain UNCW292 DQ strain UNCW277 DQ strain AY strain CBS AB strain UNCW382 DQ strain UNCW372 DQ strain CBS AB29388 strain UNCW380 DQ39356 Aphanomyces invadans EU Aphanomyces piscicida AY Aphanomyces astaci GU Aphanomyces laevis AY Aphanomyces strain APH_SAN0 FJ strain SAP4 FJ ima fungus

5 Barcode assessment within the Saprolegnia-Achyla clade Leptolegnia caudata CBS Leptolegnia caudata CBS343 Leptolegnia caudata AJ strain SAP_VIR0 FJ strain SAP_TRN0 FJ strain SAP_ZEL0_FJ strain SAP_ZLU0_FJ strain SAP_ZEL02_FJ79494 strain SAP_RIM0_FJ79490 strain SAP_ZEL03+04 FJ79492 strain SAP_BRN0 FJ strain SAP_SEC0 FJ strain SAP3_LAB0 FJ strain SAP3_ZLU0 FJ79490 Leptolegnia SAP 0.94 Leptolegnia chapmanii AJ Leptolegnia_chapmanii AJ23866 Leptolegnia sp. AJ Saprolegnia ferax AJ strain AB strain FJ strain AB Saprolegnia parasitica CBS , complete genome data strain AB7593 Isoachlya humphreyana CBS0057 (Isoachlya) «Pythiopsis cymosa» CBS26.38, misindentified (actually Protoachlya sp.) 0.86 Pythiopsis terrestris CBS Pythiopsis terrestris CBS0058 Pythiopsis intermedia CBS Pythiopsis terrestris AJ environmental clone PR4_4E_25 GU (peat bog) strain AJ (affiliation uncertain) Newbya dichotoma spec. nov Newbya apiculata AJ Aplanopsis spinosa CBS Aplanopsis spinosa CBS Aplanes treleaseanus VKM F-229 Achlya bisexualis M32705 Achlya debaryana VKM_F-904 Thraustotheca clavata CBS Thraustotheca terrestris CBS0985 Thraustotheca clavata CBS Achlya sparrowii VKM F-227 Saprolegnia Isoachlya Protoachlya Pythiopsis Newbya Aplanopsis Aplanes Achlya I Thraustotheca Achlya II environmental clone LG05- AY99696 (freshwater) strain APH_SAN0 FJ strain APH2_BRN0 FJ strain APH_VIR0 FJ Aphanomyces invadans DQ Aphanomyces invadans AF strain APH3_FAS0 FJ «Newbya pascuicola» VKM-223 (misslabelled, actually Aphanomyces) Aphanomyces cf. repetans CBS26887 HQ Aphanomyces cf. repetans CBS26886 HQ3844 Aphanomyces laevis CBS27282 HQ Aphanomyces euteiches ATCC 20684, complete genome data 0.97 environmental clone PR3_3E_94 GU (peat bog) environmental clone LG8_ AY99745 (freshwater) environmental clone LG22_02 AY99760 (freshwater) strain SAP4_ZLU0 FJ7949 Aphanomyces strain 84 AF Environmental sequences Fig. 2. Maximum Likelihood SSU rrna gene phylogenetic tree showing the position of genera within Saprolegniaceae. The tree is rooted with sequences derived from Aphanomyces species and relatives. Species and isolates that have been studied morphologically have been enclosed. Sequences marked with an asterisk correspond to misidentified strains in culture collections. pattern was contradicted by the SSU rrna gene tree. The analysis based on the SSU rrna gene (Fig. 2) respected the monophyly of all genera but did not give robust support to Leptolegnia and Pythiopsis. Genetic distances between sequences appeared visibly shorter on the tree, and distinct species within genera or between closely related genera could not be discriminated; for example, Aplanes treleaseanus and Newbya dichotoma share the same sequence at the SSU rrna level. Two supplementary deep branchings were supported by SSU rrna, the first united Saprolegnia, Fig.. (p. 442). Maximum Likelihood ITS phylogenetic tree showing the position of genera within Saprolegniaceae. The tree is rooted with sequences derived from Aphanomyces species and relatives. Species and isolates that have been studied morphologically have been enclosed. Sequences marked with an asterisk correspond to misidentified strains in culture collections. volume 5 no

6 Steciow et al. Isoachlya anisopora CBS AF9609 strain UNCW380 DQ strain UNCW382 DQ Isoachlya eccentrica AF96 Isoachlya eccentrica CBS 2.35 HQ6655 Isoachlya eccentrica CBS HQ66547 strain UNCW29 DQ strain UNCW299 DQ strain UNCW289 DQ strain UNCW298 DQ strain UNCW292 DQ strain UNCW32 DQ strain UNCW277 DQ strain AF Saprolegnia litoralis CBS 0062 HQ strain UNCW286 DQ strain UNCW284 DQ39347 strain ATCC DQ strain UNCW70 DQ strain HPJN strain UNCW62 DQ strain UNCW72 DQ strain UNCW76 DQ Saprolegnia ferax CBS HQ66542 Saprolegnia mixta CBS HQ66527 Saprolegnia unispora CBS 0066 HQ strain AF strain UNCW295 DQ Saprolegnia diclina CBS HQ Saprolegnia delica isolate CBS HQ66524 Saprolegnia parasitica CBS 387 HQ Saprolegnia parasitica CBS HQ66596 Saprolegnia parasitica CBS HQ66565 strain UNCW278 DQ Saprolegnia parasitica CBS HQ strain UNCW377 DQ strain UNCW250 DQ strain UNCW27 DQ strain UNCW28 DQ Saprolegnia parasitica CBS HQ Saprolegnia litoralis CBS HQ Saprolegnia subterranea CBS 3343 HQ Saprolegnia sp. cf. rodrigueziana CAL-20 HQ strain UNCW386 DQ strain UNCW38 DQ strain UNCW258 DQ strain UNCW257 DQ Saprolegnia monilifera CBS HQ Saprolegnia monilifera CBS HQ Saprolegnia unispora CBS HQ66552 Saprolegnia terrestris CBS 0063 HQ Scoliolegnia asterophora AF969 Scoliolegnia asterophora CBS EU54494 strain WM_2 EU54494 Isoachlya Saprolegnia Saprolegnia Scoliolegnia strain AF Protoachlya paradoxa CBS HQ Protoachlya paradoxa ATCC DQ «Pythiopsis cymosa» CBS DQ (*) Protoachlya strain UNCW244 DQ Saprolegnia monoica CBS HQ Saprolegnia monoica CBS HQ Saprolegnia_monoica AF287 strain UNCW285 DQ Saprolegnia megasperma CBS HQ66525 strain UNCW27 DQ Thraustotheca clavata AF9620 Thraustotheca clavata CBS HQ66523 Thraustotheca terrestris CBS 0985 HQ strain AF23595 strain AF Achlya debaryana VKM_F-904 Achlya americana AF2879 strain AF strain JN62755 Achlya sparrowii VKM F-227 Achlya colorata AF2877 Achlya colorata AF strain CBS HQ66520 strain CBS 33.8 HQ Achlya racemosa AF2878 Achlya radiosa AF9582 Aplanes treleaseana AF9584 Aplanes treleaseanus VKM F-229 Aplanes papillosa AF2873 Aplanes papillosa AF9580 Aplanopsis spinosa AF9589 Aplanopsis spinosa CBS Aplanopsis spinosa AF9583 Aplanopsis spinosa AF2874 Aplanopsis spinosa CBS Newbya dichotoma spec. nov 0.93 Newbya oligacantha AF2875 strain AF9593 strain UNCW36 DQ Leptolegnia caudata AF Leptolegnia caudata CBS HQ Leptolegnia caudata CBS343 Geolegnia helicoides KF Leptolegnia sp. CBS HQ strain AF strain AF strain WM3 EU54496 Thraustotheca Achlya I Achlya II Aplanes Aplanopsis Newbya Leptolegnia Geolegnia 0.83 Aphanomyces euteiches CBS HQ66529 Aphanomyces euteiches ATCC 20684, complete genome data 0.85 Aphanomyces euteiches CBS HQ66532 Aphanomyces cochlioides CBS HQ66524 Aphanomyces cladogamus CBS HQ Aphanomyces 0.0 Fig. 3. Maximum Likelihood LSU rrna gene phylogenetic tree showing the position of genera within Saprolegniaceae. The tree is rooted with sequences derived from Aphanomyces species and relatives. Species and isolates that have been studied morphologically have been enclosed. Sequences marked with an asterisk correspond to misidentified strains in culture collections. Isoachlya, Protoachlya, and Pythiopsis (bootstrap value = 0.86) and the second united this group to Newbya + Aplanes + Aplanopsis (bootstrap value = 0.8). In addition, the environmental sequence PR4_4E_25 GU appears to belong probably to either a described genus that has not been surveyed with molecular tools, or perhaps even a totally new genus. Likewise, environmental clones LG8_ (AY99745) and LG22_02 AY99760 seem to be related to both Achlya-Saprolegnia and the Aphanomyces group without any clear affinities for either of the two groups. In contrast, sequences PR3_3E_94 (GU479947) and LG05- (AY99696) branched within the Aphanomyces clade. The phylogeny resulting from the LSU rrna sequences (Fig. 3) showed a somewhat intermediate situation: some genera did not appear monophyletic (i.e. Saprolegnia, Isoachlya) or were not strongly supported (Aplanopsis). However, this analysis supported a robust (bootstrap = 0.87) branching between Thraustotheca, Aplanes, and Aplanopsis, a relationship that was not recovered in analyses based on the two other markers used (ITS and SSU rrna). The resulting consensus tree had the genera Leptolegnia, Geolegnia, and undescribed genus SAP branching together, and also a group composed by Aplanopsis, Achlya, and Thraustotheca as the most basal known members of 444 ima fungus

7 Barcode assessment within the Saprolegnia-Achyla clade the group. Newbya and Aplanes occupied an intermediate position, while Saprolegnia, Isoachlya, Protoachlya, and Pythiopsis were resolved as the most derived (Fig. 4). The genus Achlya divided into two relatively distinct groups, named here as Achlya I (containing A. colorata and A. radiosa), and Achlya II (for A. bisexualis and related strains). Taxonomic reliability in GenBank There were two types of errors in sequence assignment in this group of fungi in GenBank, taxonomic mistakes and incorrectly named material. The first type of mistake (referred to as type hereafter) is caused by lumping different genera together by placing them into Achlya, Saprolegnia or Pythiopsis. This concerns, respectively, the genera Scoliolegnia, Leptolegnia, SAP, part of Isoachlya, and Protoachlya all into genus Saprolegnia, and part of Isoachlya into Pythiopsis and Aplanes, and Newbya and Aplanopsis into Achlya. A second type (type 2) came from incorrect naming of strains, and occurs even when strains were apparently ordered from a public culture collection. Results of this investigation are presented in Table 2. We also faced the same problems with the strains we ordered: Pythiopsis cymosa (CBS 26.38) was actually a Protoachlya, Thraustotheca clavata (CBS ) was Leptolegnia caudata, and Newbya pascuicola (the type species of Newbya), now lost in culture, possibly through contamination with an Aphanomyces (Fig. 2). TAXONOMY Achlya I Achlya II, Thraustotheca «SAP» Aphanomyces Saprolegnia Pythiopsis Protoachlya Isoachlya Newbya Aplanopsis Aplanes Leptolegnia, Geolegnia Fig. 4. Consensus tree of the Maximum Likelihood ITS, SSU and LSU rrna phylogenetic trees showing the position of genera within Saprolegniaceae. One strain isolated in the course of this study showed unique morphological features different from those of closely related species (Dick 200) justifying its description as a new species. A detailed morphological description is provided below. Markers ITS and LSU rrna successfully discriminated it from closely related species (Figs, 3), whereas the SSU rrna gene was not variable enough to discriminate it from Achlya apiculata (AJ238656; syn. Newbya apiculata), Achlya spinosa (CBS ; syn. Aplanopsis spinosa), and Aplanopsis spinosa (CBS ). Newbya dichotoma Steciow, Lara & Belbahri, sp. nov. MycoBank MB80867 (Figs 5 6) Etymology: The name refers to the extremely well developed hyphae that are bifurcate (dichotomously branched), one or more times. Diagnosis: Mycelium densum, cultura in seminibus Cannabis sativa ca cm diam. Hyphae ramosa, pleraque µm late ad basim. Zoosporangia parcus in culturis juvenilibus filiformia vel fusiformia, 800( 800) (9 )24 72 µm, basipeta vel cymosa. Ejecto sporarum pro genus typica, cystae globosi 0 2 µm. Gemmae frequentis. Oogonia copiosa, apiculata, sphaerica, pyriformia vel doliiformia, vel paulo abnormia (27 )45 87( 46) µm. Paries oogoni foveatus, laevis; ramulus lateralibus vel terminalibus provenientia, 9 63( 437) µm. Oosporae subcentrici; ( )2 5( 7) per oogonium, (9 )25 3( 4) µm. Ramulus antheridialis ramosus, monoclina, diclina, et androgina. Type: Argentina: Neuquén Province, Chimehuín River, at 7 km from Junin de los Andes town, ( S O), on floating organic matter, in water associated with aquaculture ponds where salmonids (Onchorhynchus mykiss, and Salmo trutta) and atherinopsids (Patagonian silverside: Odonthestes hatcheri) are raised, May 200, M. M. Steciow (LPS holotype; LPSC 877 ex-type culture). Description: Monoecious. Two-week-old pure cultures on hemp seeds cm diam. Mycelium dense, extensive; principal hyphae slender or stout moderately branched, finishing in characteristic dichotomous branching near apices; µm wide at the base. Gemmae sparse or abundant with the age of the water culture; cylindrical, fusiform, irregular, or branched; terminal or intercalary, single or catenulate, developed at the end of the hyphae functioning as zoosporangia. Zoosporangia sparse, slender; filiform, or fusiform; often tapering to the elongate apex; frequently straight, curved, bent or sinuous; (80 )50 800( 300) µm; renewed sympodially or in cymose disposition, rarely in basipetalous succession. Zoospores monomorphic; discharge and behaviour achlyoid; primary spore cysts, µm diam. Oogonia variable in abundance or abundant with the age of the culture; lateral or terminal, occasionally intercalary, single or catenulate; apiculate, often spherical, subglobose, pyriform, or very rarely oval or irregular, occasionally doliiform, very rarely filiform; oogonia very variable in size and number with the age of the culture, often µm diam in average when small and developing as normal ones, and predominantly 45 90( 0) µm diam when bigger. Oogonia with oospores that can mature or not inside and develop into a lower number of oospores, often unique, mainly with a great size; volume 5 no

8 ART I CLE Steciow et al. 446 Fig. 5. Morphological features of Newbya dichotoma (LPS 47444) A. A young water culture with zoosporangia at the tips of the mycelium. B. A 2-wk-old hemp seed colony in water culture. C D. Characteristic dichotomous branching of hyphae repeated in one or several times. E. Zoosporangium filiiform, tapering to the elongate apex. F. Zoosporangium showing typical achlyoid discharge. G. Ooogonia very variable in size, maturity and number of oospores inside, with the age of the culture. H. Oogonial stalks variable in length, curved, bent, coiled or straight, often branched. Bars: C = 200 µm, D E, G = 00 µm; F, H = 25 µm. Fig. 6. Morphological features of Newbya dichotoma (LPS 47444) A B. Mycelium with immature and some mature oogonia, often apiculate, on variable oogonial stalk, straight, bent or curved. C. Detail of a mature apiculate oogonium with a monoclinous antheridial branch. D. Immature oogonium with diclinous branches. E H. Mature oogonia apiculate, spherical to subglobose, showing, in detail, the subcentric oospores type I, variable in number, size and maturity. Bars: A D = 25 µm, E H = 5 µm. very frequent proliferation of immature and mature oogonia. Oogonial wall smooth, slender, (very rarely with a lateral papillate projection); unpitted, or pitted only under point of attachment of antheridial cells; inner surface occasionally irregular. Oogonial stalks variable in length, usually 0.5 4( 9) times the diameter of the oogonium; slender and short or stout and longer; often curved, bent, twisted and often once-several times coiled, rarely straight; often branched. Oospores almost always maturing inside normal oogonia, or not maturing inside some of the greatest or abnormal ones; subcentric type I; spherical or ellipsoidal, or irregular when immature; ( )2 5( 7) per oogonium, filling it or not filling the greatest or abnormal oogonia with often a greater normal-mature or abnormal not maturing oospore inside; (20 )25 35 µm diam inside smaller oogonia, and (35 ) 50 70( 80) µm diam inside greater oogonia. Antheridial branches usually abundant; mainly monoclinous, often diclinous, rarely androgynous; slender, irregular; abundantly branched; persisting. Antheridial cells simple, short; tubular to irregular; persisting; laterally appressed; fertilization tubes not observed. Notes: The genus Newbya s. str. includes species previously named Achlya and now separated as: N. apiculata, N. braziliensis, N. curvicollis, N. megasperma, N. oblongata, N. oblongata var. gigantica, N. oligacantha, N. pascuicola (type species), N. polyandra, N. recurva, N. spinosa, and N. stellata (Spencer et al. 2002). Newbya dichotoma resembles N. apiculata. Both species have smooth oogonia that are spherical or apiculate, borne on slender or stout, straight, curved, bent, twisted or more characteristically coiled oogonial stalks, one to several times. The oogonial stalks of N. apiculata are branched, and the antheridial branches are predominantly monoclinous. ima fungus

9 Barcode assessment within the Saprolegnia-Achyla clade Newbya apiculata forms centric or preferentially subcentric oospores, reaching (20 )35 40( 48) µm diam. In contrast, in N. dichotoma, the oogonia contain a more reduced number of only subcentric oospores ( 7 vs. 28 in N. apiculata), which are often variable in size and number within the same thallus. Oogonia containing only a few oospores predominate: one, two, or commonly up to four oospores, which can reach a relatively large diameter: 50 70( 80) µm diam. Newbya dichotoma also develops a considerable number of oogonia containing 3 5( 7) smaller oospores, that reach only µm diam (Figs 5 6). Discussion A major problem in the taxonomy of Saprolegniales that is encountered, especially in the Saprolegnia-Achlya clade, is the lack of clear and unique synapomorphies that can define genera. Rather, genera are defined by a combination of traits, which seem to revert frequently to ancestral states or even disappear along evolution (Dick et al. 999, Dick 200). A good example is the genus Geolegnia, represented here by G. helicoides, that has lost the entire flagellar apparatus in all life-stages seen in Leptolegnia-like ancestors (Steciow et al., 203). An illustration of these changing characters is given in Table 2. It is, for instance, hazardous to infer the morphology of the ancestor of the whole clade. As in better known fungal groups such as the basidiome-forming Agaricomycotina, the combination of various traits, rather than single ones, is the only possibility to identify species morphologically (Zmitrovich & Wasser 20). Some other traits that can possibly be discriminating for the different subgroups can be inferred by searching into the metabolism of these organisms and their enzymatic machinery. For instance, members of the genus Leptolegnia are known to degrade chitin (Hochwimmer et al. 2009), being important mosquito pathogens (Pelizza et al. 20). The related Geolegnia helicoides has also been isolated from mosquitos (Steciow et al. 203) and the equally related undescribed genus SAP- (Wolinska et al. 2009) on cladocerans. It is therefore likely that the ability to use chitin is a common feature of this group (Fig. 4). Other biochemical characteristics, not necessarily directly related to pathogenesis, might be found in other clades, and a good barcoding strategy appears to be the most reasonable alternative way to identify these organisms. For this purpose, we tested the resolution power of three common barcoding genes used for many protists and fungi (Pawlowski et al. 202). ITS clearly had the best potential for species barcoding purposes, as it separated closely related species within genera most efficiently (Robideau et al. 20). ITS has, however, been shown not to be reliable for other (presumably older) clades of oomycetes such as Pythium s. lat. (Levesque & DeCock 2004), but it is perfectly suited in the context of the Saprolegnia-Achlya clade. Deeper nodes appear most often unresolved by ITS, and there ribosomal genes are more useful and have been recommended for general oomycete phylogeny reconstruction (Riethmüller et al. 999, Leclerc et al. 2000, Lara & Belbahri 20). As the SSU rrna gene is often used in environmental DNA surveys, it can be a potential source of discovery for new species by revealing the phylogenetic position of organisms whose existence was unsuspected (Fig. 2) and which can potentially be emerging parasites, or possibly also important ecological actors, as the ecological role of free-living oomycetes remains a largely uncharted territory (Lara & Belbahri 20). Our study clarifies the phylogenetic relationships within the Saprolegnia-Achlya clade based on a combination of morphological features and a consensus tree derived from three phylogenetic markers. We demonstrate that the divisions between genera proposed by Spencer et al. (2002) were justified and further confirm that this clade is more genusrich than initially proposed (Spencer et al. 2002, Hulvey et al. 2007). The formal description of Newbya dichotoma provided in this study illustrates the need for an effort towards culturing new strains and describing them morphologically prior to their genetic characterisation. Still, some genera probably associated with this clade have not been surveyed, such as Brevilegnia, Calyptralegnia, Dictyuchus, and Scoliolegnia, and they need to be targeted by these same markers in future studies. Here, we have, however, laid the basis of a taxonomic framework for the efficient recognition and description of pathogens within this clade of organisms of major economic and environmental concerns. A good reference database is logically the most basic prerequisite for an effective barcoding strategy, the other being good marker genes that allow species discrimination (Hebert et al. 2003). It emerges from our survey that an uncritical use of GenBank as a reference database can be a source of mistakes that may only amplify the problems when taken as a basis for further studies. Type mistakes (wrong taxonomy) might seem less problematic than type 2 (incorrect identifications); they are, however, a problem for non-specialists who want to use oomycete sequences as references in their phylogenies, thus encountering pervasively para/polyphyletic genera. These mistakes can be a problem in environmental DNA diversity surveys. Type 2 mistakes are of course of major concern when unknown strains have to be identified molecularly, and can lead to misinterpretations that can bear heavy consequences for wildlife management or economic issues. Therefore, we recommend that mistakes that have been identified are corrected, and further that cultures made available from culture collections are checked for such inconsistencies. AcknowledgEments M.M.S. thanks CONICET (PIP 422) and UNLP (Proyect N/ 566) for financial support. This project also received funding from the European Union s Seventh Framework Programme for research, technological development and demonstration under grant agreement no (to L.B.). This study was also supported by the SwissBOL project, financed by the Swiss Federal Office for the Environment (Grant holder L.B.), the Swiss State Secretariat for Education and Research (L.B. grant reference: SER no. C09.039), and the European Union for the COST action FP080 Established and Emerging Phytophthora: Increasing Threats to Woodland and Forest Ecosystems in Europe. volume 5 no

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